Abstract

The objectives of this study were to identify potential alterations in gene expression of melanocortin-4 receptor (MC4-R), proopiomelanocortin (POMC), and Agouti-related protein (AgRP) in mouse hypothalamus under a chronic peripheral infusion of leptin or at early (8 weeks) and advanced (16 weeks) phases of diet-induced obesity. Control or diet-induced obesity mice (8 or 16 weeks of high-fat diet) were either treated or not treated with leptin. Metabolic features were analyzed and expression of the genes of interest was measured by quantitative reverse transcriptase-PCR (RT-qPCR) and western blot. We reported that in control mice, but not in obese mice, leptin infusion induced an increase in POMC mRNA level as well as in MC4-R mRNA level suggesting that leptin could act directly and/or through α-melanocyte-stimulating hormone (α-MSH). This hypothesis was reinforced after in vitro studies, using the mouse hypothalamic GT1-7 cell line, since both leptin and Norleucine4, D-Phenylalanine7-α-MSH (NDP-α-MSH) treatments increased MC4-R expression. After 8 weeks of high-fat diet, nondiabetic obese mice became resistant to the central action of leptin and their hypothalamic content of POMC and AgRP mRNA were decreased without modification of MC4-R mRNA level. After 16 weeks of high-fat diet, mice exhibited more severe metabolic disorders with type 2 diabetes. Moreover, hypothalamic expression of MC4-R was highly increased. In conclusion, several alterations of the melanocortin system were found in obese mice that are probably consecutive to their central resistance to leptin. Moreover, when the metabolic status is highly degraded (with all characteristics of a type 2 diabetes), other regulatory mechanisms (independent of leptin) can also take place.

Introduction

Obesity is a major cause of disease in western populations with increased morbidity and mortality risks. It is associated with the metabolic syndrome characterized, in particular, by type 2 diabetes and cardiovascular diseases. Several hormonal signals target the hypothalamus known to be the centre of control energy homeostasis, hunger, and satiety. In the melanocortinergic system, MC3-R and MC4-R play a major role in controlling energy storage and food intake, respectively, through binding of α-MSH which originated from the cleavage of proopiomelanocortin (POMC) (1).

MC4-R is expressed within several regions of the brain, including thalamus, hippocampus, spinal cord, cortex, and hypothalamus where it plays a fundamental role in the control of feeding and body weight homeostasis (2). MC4-R −/− mice are obese, hyperphagic, and have an increased somatic growth (3). In human various mutations of the MC4-R have been associated with an obese phenotype (4). Interestingly, there is an endogenous antagonist for these receptors, the Agouti-related protein (AgRP) (5). Transgenic mice that overexpress AgRP are hyperphagic and develop an early obesity associated with hyperinsulinemia and fasted hyperglycemia (6).

In the hypothalamus, the arcuate nucleus contains the first-order neurons that are sensitive to several hormones such as leptin and insulin present in blood and to nutrients such as glucose and lipids. These neurons project to other hypothalamic regions such as paraventricular, dorsomedian, and ventromedian nuclei which contain second-order neurons expressing, in particular, MC4-R and/or MC3-R (7). Leptin, a polypeptide secreted by adipose tissue, inhibits food intake and causes a reduction in body weight (8,9) after it crosses the blood-brain barrier by a saturable transport system (10) and the melanocortin system is involved in this effect (11). At the arcuate nucleus level, leptin is known to modulate both POMC and AgRP neurons by increasing POMC expression and decreasing AgRP expression (11,12). The nonselective melanocortin receptor antagonist SHU9119 suppresses the inhibitory effect of leptin on food intake and body weight in rats (13). All these data suggest that the central effects of leptin may involve changes not only in the expression of genes encoding MC-R ligands but also in the expression of genes encoding the melanocortin receptors (14). Therefore, the aim of this work was to study the alterations in the expression of the genes encoding different components of the melanocortinergic system in mouse in two conditions where plasma leptin levels are modified and thus where its central action would be altered: first, in diet-induced obesity at an early phase or at a later phase when the obese phenotype has evolved to the metabolic syndrome; second, in standard diet conditions under a chronic leptin infusion. In parallel, in vitro studies have been performed on the hypothalamic cell line GT1-7 to complete some data obtained by in vivo studies.

Animals

All procedures were performed with the approval of the Regional Committee of Ethic for Animal Experiments. Four-week-old male C57Bl/6J mice were purchased from Harlan (Le Marcoulet, France) and housed at 21 °C with normal light-dark cycle and free access to water and food. After 1-week acclimatization (with standard chow), mice were randomized to perform two series of experiments.

In a first series of experiments, mice were divided in two groups. One group (control) had free access to standard chow A04 (UAR, Villemoisson-sur-Orge, France) and another group (DIO) to a high-fat (HF) diet, from Harlan-Teklad (Madison, WI) for 8 weeks (8w-control or 8w-DIO) or 16 weeks (16w-control or 16w-DIO). This HF diet contains 36.1% fat vs. 3.1% in the standard diet and has been previously described (15). Food intake and body weight were measured every 2 days.

In a second series of experiments, mice were divided in two groups fed either with standard or HF diet for 7 weeks. Afterward, both groups were subdivided in two subgroups. Each subgroup received an Alzet osmotic pump (Model 1007D, Alza, Palo Alto, CA) placed (for 1 week) in the peritoneal cavity under anesthesia. Each pump delivered either 10 μg of recombinant ovine leptin (OvLept, (16)) per day in a total volume of 12 μl of phosphate buffer saline 10 mmol/l pH 7.4 used as diluent or 12 μl of diluent alone. It has to be noted that ovine leptin shows the same biological activity compared to murine leptin (Gertler, personal communication). Groups were named sham-control and control + OvLept or sham-DIO and DIO + OvLept.

At the end of each experiment, the mice were killed by cervical dislocation after a 6-h fasting to ensure that mice were at the same postabsorptive stage. The brain was removed and the whole hypothalamus was rapidly dissected out and frozen in liquid nitrogen. The limits of the hypothalamus for dissection were the optic chiasma at the anterior border, the mammillary bodies at the posterior border, and, on both lateral sides, the hypothalamic sulci. The tissue was finally cut dorsally at 2 mm from the ventral face. The blood was collected for measurement of glucose (OneTouch Ultra; Lifescan France, Issy-les-Moulineaux, France), murine leptin (EIA kit BioVendor; AbCys, Paris, France), murine insulin (EIA kit Crystalchem; Chicago, IL), and triglycerides (PAP150 kit; BioMérieux, Marcy-l'Etoile, France).

Cell culture

The mouse hypothalamic GT1-7 cell line was used to study the effects of hormonal treatment on the expression of the MC4-R encoding gene. Two days before treatment, cells were plated on 6-well dishes at 200,000 cells per well, in 25 mmol/l glucose Dulbecco's modified Eagle's medium containing penicillin (100 U/ml), streptomycin (0.1 g/l), and glutamine (100 U/ml) and supplemented with 10% fetal calf serum, at 37 °C under 5% CO2. The day before treatment the medium was replaced by complete Dulbecco's modified Eagle's medium supplemented with 1% fetal calf serum. Treatments were performed during 3–24 h in complete Dulbecco's modified Eagle's medium supplemented with 1% fetal calf serum, in the presence of either leptin or NDP-α-MSH.

Quantitative reverse transcriptase-PCR

Quantitative reverse transcriptase-PCR (RT-qPCR) was used to study the expression of the different target genes. Total RNA was extracted from either whole hypothalami or GT1-7 cells using TRIZOL (Invitrogen) according to the manufacturer's protocol. All samples were treated by DNaseI (Invitrogen) before the reverse transcription. First-strand cDNAs were prepared using 1 μg RNA and SuperScriptII Reverse Transcriptase (Invitrogen) in the presence of random hexamer and oligo(dT) primers (Promega, Charbonnières-les-Bains, France). The qPCR reactions were performed using the Light Cycler Fast Start DNA Master SyBR Green I kit (Roche, Meylan, France) in the presence of specific primer pairs which were selected to amplify small fragments (100–200 bp) (Table 1). PCR products were checked for specificity by measuring their melting temperature. Samples (in duplicate) were quantified by comparison with a standard curve obtained by dilutions of purified specific cDNAs.

Table 1. Primers used for the qPCR

Western blotting

Ten micrograms of proteins, prepared from whole hypothalami or GT1-7 cells (TRIZOL extraction), were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis 10% and transferred to a polyvinylidene fluoride membrane (Immobilon-P transfer membrane, Millipore, Saint-Quentin-en-Yvelynes, France). Immunoblotting was performed using rabbit antibodies directed against mouse MC4-R (1,000-fold dilution), monoclonal antibodies against β-actin (10,000-fold dilution), and mouse β-tubulin (1,000-fold dilution). The Envision System Labelled-Polymer-HRP antirabbit (DakoCytomation, Trappes, France) was used as secondary antibody. Blots were revealed using the ECL-Plus Western Blotting Detection System (Amersham Biosciences, Orsay, France). For MC4-R the band obtained was detected at 55 kDa which might correspond to a glycosylated form. The specificity of the MC4-R antibodies (no crossreactivity with MC3-R) was controlled by using proteins obtained from HEK-293 cells stably expressing either MC4-R (17) or MC3-R (18).

Statistics

Two-way ANOVA (diet × leptin treatment) was used to analyze the effects on mRNA expression (Statview software) and significant effects were then ascertained with post hoc Fisher least square difference test for all pair comparisons. Other statistical analyses were performed using one-way ANOVA followed by post hoc testing with Fisher's protected least square difference test. Differences were considered significant at P < 0.05.

Results

Metabolic characteristics

DIO mice became obese after 5 weeks of HF diet with a weight at least 20% above control mice (Figure 1). After 8 and 16 weeks of HF diet, DIO mice showed a weight 47 and 80% higher than control mice, respectively, corresponding to a dramatic increase in weight gain (Table 2 and Figure 1). However, the DIO mice did not ingest higher amounts of diet compared with control mice (data not shown) but their average energy intake was higher than in control mice (13.1 ± 0.3 vs. 10.5 ± 0.3 kcal/day/mouse).

Figure 1. Weight curves of the mice. Change in weight of mice during 16 weeks of control (16w-control) or high-fat (16w-DIO) diet. *P < 0.05 compared to 16w-control mice.

The plasma leptin levels, reflecting the level of adiposity, were dramatically increased by 12.6-fold in 8w-DIO and 6.1-fold in 16w-DIO compared to control mice at the same age. The difference was less pronounced in 16w-DIO mice because of a concomitant twofold increase in the leptin plasma level that occurred in control mice with ageing (Table 2). After a 6-h fast, corresponding to a postabsorptive stage, plasma insulin in 8w-DIO and 16w-DIO was increased by 3.5- and 4.0-fold, respectively, compared to control mice at the same age (Table 2). Both 8w-DIO and 16w-DIO mice were hyperglycemic after a 6-h fast, compared to control mice (Tables 2 and 3). But after a 16-h fast, 8w-DIO mice did not exhibit hyperglycemia contrary to 16w-DIO mice (Table 2).

Control + OvLept mice exhibited a significant loss of weight (−7.7%) (Table 3), which correlates with the loss of fat mass already reported (15), and their plasma insulin decreased by 70% (Table 3). DIO + OvLept mice did not show any weight change when compared to Sham-DIO mice, which indicates central leptin resistance, and their plasma insulin was also decreased but only by 40%. The plasma triglyceride levels were increased by 1.9-fold in sham-DIO mice (1.0 ± 0.2 g/l) compared to sham-control (0.53 ± 0.05 g/l). This level was decreased by 36% in control + OvLept mice (0.34 ± 0.05 g/l) and by 20% in DIO + Ovlept mice (0.8 ± 0.1 g/l). Altogether, these results indicate that, at this stage of obesity, the peripheral tissues did not display the same degree of leptin resistance that was already firmly established at the central level.

Hypothalamic levels of mRNA encoding the melanocortinergic genes

By RT-qPCR, no significant differences were observed in the level of MC4-R mRNA in the hypothalamus of 8w-DIO mice when compared to 8w-control mice (Figure 2a). On the contrary, a 1.9-fold increase in MC4-R mRNA level was found in 16w-DIO mice vs. 16w-control mice (Figure 2a). Western blot analysis showed that the MC4-R protein levels were also strongly increased in 16w-DIO mice (Figure 2b). The levels of both POMC and AgRP mRNA were decreased by ∼35% in 8w-DIO mice (Figure 2c,d) but tended to increase, even though not significantly, in 16w-DIO mice (Figure 2c,d).

Figure 2. Hypothalamic expression of melanocortinergic genes in control and DIO mice. (a) MC4-R measurement by RT-qPCR. (b) Western blot analysis was performed using proteins from whole hypothalami of 16w-control and 16w-DIO mice and antibodies against MC4-R or β-actin. (c) POMC, and (d) AgRP measurement by RT-qPCR. Results of RT-qPCR are expressed as the ratio between values obtained with the gene studied and value obtained with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (GAPDH expression was not modified by aging, high-fat feeding, or leptin treatment). Each value represents the mean ± s.e.m. of six to nine different mice.

Interestingly, we found a twofold increase in MC4-R mRNA level between 8w- and 16w-control mice, which was even more pronounced in DIO mice (Figure 2a). Similarly, at 16 weeks, the levels of POMC and AgRP mRNA were both increased in control and DIO mice compared to 8 w-control and 8w-DIO (three- and sixfold, respectively), as shown in Figure 2c,d, indicating an age-related variation.

Effect of in vivo and in vitro leptin treatment

RT-qPCR showed a 1.7-fold increase of MC4-R mRNA level in the hypothalamus of control + OvLept, compared to sham-control mice (Figure 3a), while it remained unchanged between DIO + OvLept and sham-DIO mice (Figure 3a).

Figure 3. Effect of a 1-week leptin infusion on the hypothalamic expression of melanocortinergic genes. (a) MC4-R, (b) POMC, and (c) AgRP measurement by RT-qPCR. Results are expressed as fold stimulation over the sham-control mice. Each value represents the mean ± s.e.m. of six to nine different mice.

While a 1.4-fold increase in the level of hypothalamic POMC mRNA was measured in control + OvLept compared to sham-control mice (Figure 3b), no variation could be found between DIO + OvLept compared to sham-DIO mice (Figure 3b) confirming the loss of sensitivity to leptin in DIO mice. As for AgRP, its mRNA level remained unchanged (Figure 3c).

GT1-7 cell line was used to determine whether leptin could directly regulate the expression of the MC4-R encoding gene. Contrary to the in vivo results, no difference in the MC4-R mRNA level was observed (Figure 4a) following a 24-h treatment with 10,000 ng/ml of OvLept. Stimulations with lower concentrations of leptin (100 or 1,000 ng/ml) or for shorter periods of time (3–16 h) remained without any effect on MC4-R mRNA levels (data not shown). Interestingly, after the 24-h treatment with 10,000 ng/ml of OvLept, a threefold increase of MC4-R protein level (Figure 4b) could be detected by western blotting. The good correlation between the increase in POMC and MC4-R mRNA levels in response to leptin treatment observed in vivo, prompted us to check for any effects of α-MSH on the expression of MC4-R in GT1-7 cells. Twenty-four hour stimulation with 0.1 nmol/l of NDP-α-MSH resulted in a 1.7-fold increase in MC4-R mRNA (Figure 4a) and protein levels (Figure 4c). It has to be noted that GT1-7 cells did not express detectable level of POMC mRNA both in control and leptin-treated cells (data not shown).

Figure 4. In vitro studies of the expression of the MC4-R encoding gene. GT1-7 were treated for 24 h with either 10,000 ng/ml of OvLept or 0.1 nmol/l NDP-α-MSH. (a) corresponds to measurement by RT-qPCR and results are expressed as fold stimulation over the control (mean ± s.e.m. of at least three different experiments). (b) and (c) correspond to western blot analyses performed in the presence of antibodies against either MC4-R or β-tubulin.

Discussion

After 8 weeks of HF diet, DIO mice displayed a severe obesity associated with hyperinsulinemia, hypertriglyceridemia, and hyperleptinemia. A postabsorptive (6-h fast) hyperglycemia was also observed but not after 16 h of fasting. This would indicate that these mice were insulin resistant but did not exhibit type 2 diabetes. After 16 weeks of HF diet, mice displayed type 2 diabetes with hyperglycemia after 16 h of fasting. The leptin treatment, which caused a loss of weight in control mice, had no effect on DIO mice after 8 weeks of HF diet, which confirms that central leptin resistance was rapidly acquired (15). The development of leptin resistance at the central level in obese mice is well documented (19,20,21,22). In particular, intracerebroventricular injection of leptin in glucose-intolerant and insulin-resistant DIO mice after 20 weeks of HF diet did not induce reduction of food intake, body weight, or hypothalamic c-Fos expression because of the lack of responsiveness of the hypothalamus to this hormone (22).

The hyperinsulinemia observed in sham-DIO mice was reversed in DIO + OvLept mice. Indeed, leptin is well known to decrease the release of insulin by human pancreatic islets in vitro (23). A moderate decrease of plasma TG concentration was observed in DIO + OvLept mice compared to control + OvLept. Our results are in agreement with those of Huang et al. (24) who demonstrated hepatic leptin resistance as a potential mechanism contributing to dyslipidemia in obesity. Our results showed that several peripheral tissues retained part of their responsiveness to leptin when obesity is progressing, contrary to brain tissue.

Leptin induced an increase in the expression of POMC encoding gene with no change in AgRP gene expression in control + OvLept mice. This increase in hypothalamic POMC mRNA expression reflects the anorexigenic effect of leptin. In normal physiological conditions, leptin is known to increase the expression of the POMC encoding gene and then the release of α-MSH in the first-order neurons of hypothalamic arcuate nucleus (11). Interestingly, in control mice, leptin was also able to enhance the level of the MC4-R encoding mRNA which is expressed mainly in second-order neurons. This raises the question of a direct or indirect effect of leptin on the expression of this gene.

A treatment of GT1-7 cells by leptin had no direct effect on MC4-R mRNA but increased protein levels possibly through an increase of the stability of the protein contrary to treatment with NDP-α-MSH that increased both MC4-R mRNA and protein levels. This positive regulation of the expression of a G-protein receptor encoding gene by its natural agonist has been previously described in our laboratory for the melanocortin receptor MC2-R (25,26,27,28). We have postulated a direct effect of leptin on MC4-R protein expression due to the fact that GT1-7 cells express only low amount of POMC mRNA. In these cells, the effect of leptin could not act through melanocortins but we cannot exclude that leptin may affect the expression of other genes that, in turn, increase the MC4-R protein expression. We can then hypothesize that, in normal physiological conditions, leptin could positively regulate the expression of MC4-R both through a direct effect and indirectly through the enhancement of POMC/α-MSH release. This mechanism would also amplify the anorexigenic response to α-MSH. Moreover, the age-related increase in circulating leptin, already reported by Ahren et al. (29), and in hypothalamic POMC and MC4-R expression levels, as observed herein in older control mice, reinforced the hypothesis of a regulation of MC4-R expression by leptin through an increase of POMC expression.

In the early phase of the development of obesity, corresponding to a prediabetic stage, we did not find any significant alterations in the level of hypothalamic MC4-R gene expression in 8w-DIO mice, but the expression of both POMC and AgRP encoding mRNA was rather decreased. Schwartz et al. (11) observed that decrease in POMC expression in the arcuate nucleus of ob/ob mice could be restored to normal after treatment with leptin. Wang et al. (30) found decreased levels of AgRP mRNA in the arcuate nucleus of mice fed with a high saturated fat diet for 1 and 7 weeks compared to different groups of mice fed with low-fat diet. Altogether, these results could be explained by the progression of hypothalamic leptin resistance. Our results are not in accordance with those of Harrold et al. (31) who reported a diminution of MC4-R gene expression in hypothalamic nuclei isolated from diet-induced obese and nondiabetic rats fed during 8 weeks with a moderate fat-rich diet. The divergence is probably due to variations between animal species, composition of the diet, or length of the feeding.

In 16w-DIO mice, an overexpression of the MC4-R gene was observed although the mice were resistant to the action of leptin and the level of POMC mRNA was similar to that of 16w-control mice. MC4-R is expressed in several nuclei of the hypothalamus and, in particular, in the paraventricular nucleus which is innervated by POMC neurons and plays an important role in the control of food intake. Our results in the whole hypothalamus are in accordance with those obtained by Enriori et al. (22) using in situ hybridization. They showed a significant increase of MC4-R expression in the paraventricular nucleus of glucose-intolerant and insulin-resistant mice after a 20-week HF diet. In our model, we hypothesize that the variation of expression of both endogenous agonist and antagonist allows first the regulation of food intake and consecutively of the metabolic parameters. Later, the increased expression of MC4-R encoding gene may amplify the anorexigenic response to α-MSH. Indeed, a direct and more prolonged action of agonists has been shown in DIO compared to low-fat fed mice (32). Thus, some authors considered that the sustained responsiveness was due to the overexpression of hypothalamic MC4-R gene induced by a hypersensitivity of the melanocortinergic system in response to stimulation (22). Our data suggest that in this late phase of extreme obesity, where mice presented all characteristics of the metabolic syndrome with type 2 diabetes, other regulatory mechanisms independent of leptin and POMC are activated to increase the expression of the receptors to improve their metabolic status. This is in agreement with the increase of MC4-R expression observed in several hypothalamic nuclei of obese fa /fa Zucker rats despite the lack of functional leptin receptor in these animals (31). The authors suggest that the increase in MC4-R density reflects receptor upregulation secondary to a reduction in α-MSH release, consistent with increased hunger (31).

Other factors could be involved in the control of food intake at the central level such as glucose (33,34) or fatty acids (35,36) plasma levels of which are high in DIO mice. Interestingly, POMC neurons are glucose-responsive neurons (34) and glucose was shown to regulate the expression of the POMC encoding gene in N-43/5 POMC-expressing neuronal cell line (37). It was suggested that malonyl-CoA, an intermediate in fatty acid biosynthesis, could control food intake and energy storage via downregulation of AgRP expression and upregulation of POMC expression within the hypothalamus (36). The high concentration in plasma TG that we observed in 8w-DIO mice might as well reflect an increase of fatty acid synthesis leading to decrease in AgRP gene expression. Some authors proposed that changes in MC4-R availability, with altered nutritional status, were mediated solely by changes in AgRP (38) suggesting that AgRP may regulate the activity of the MC4-R and thus the tonic restraining effect of the melanocortin axis on food intake and body weight. However, this regulation did not prevent the development of diet-induced obesity and its complications, as observed in other studies (21).

In conclusion, we demonstrated herein that leptin could positively regulate the level of MC4-R protein both directly and indirectly through its stimulatory effect on POMC expression. This partly explains the central hypophagic actions of leptin in normal metabolic conditions. In the early phase of diet-induced obesity, the central resistance prevents the regulation induced by leptin on POMC gene leading to a stabilization of MC4-R mRNA expression. Later, when the metabolic syndrome with type 2 diabetes is established, there was an overexpression of MC4-R involving regulatory factors other than leptin or POMC. We could hypothesize that all these compensatory modifications would enhance the responsiveness to the agonist to normalize weight and metabolic status.

Acknowledgment

J.G. was supported by grants from the Société Française de Nutrition and from the Institut Danone. J.T. was supported by grants from the French Ministère de l'Enseignement Supérieur et de la Recherche and from the Fondation pour la Recherche Médicale (Paris). A.B. was supported by a grant from the Fondation pour la Recherche Médicale (Paris). The research project was partly supported by Agence Nationale pour la Recherche (ANR 1.14, Nutrisens project). We thank Dr Gertler, Rehovot, Israel for the gift of ovine leptin and Alexandra Vandermoeten, Annie Desenfant, and Marion Apffel from Animalerie Lyon Est Conventionnelle et SPF (Université Claude Bernard Lyon 1, Institut Fédératif de Recherche 62 Lyon Est) for technical assistance and animal care. We are grateful to Dr Derrien for reviewing the English manuscript.